thin layer chromatography to identify compounds

Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids.

A small drop of the mixture is placed on the base line of the thin layer plate, and similar small spots of the known amino acids are placed alongside it. The plate is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5.

The left-hand diagram shows the plate after the solvent front has almost reached the top. The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin.
 The stationary phase - silica gel

Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups.
So, at the surface of the silica gel you have Si-O-H bonds instead of Si-O-Si bonds. The diagram shows a small part of the silica surface.
The surface of the silica gel is very polar and, because of the -OH groups, can form hydrogen bonds with suitable compounds around it as well as van der Waals dispersion forces and dipole-dipole attractions.
The other commonly used stationary phase is alumina - aluminium oxide. The aluminium atoms on the surface of this also have -OH groups attached. Anything we say about silica gel therefore applies equally to alumina.

Characterstics of different membranes

Characteristics	MF	UF	NF	RO
Membrane pore size (microns)	10-1 to 10	10-2 to 10-1	10-3 to 10-2	10-3 to10-3
Operating pressure (psi.)	1 to 25	10 to 200	300	1500
Porosity (%)	~70	~60	~50	~50
Structure	Several types	Asymmetric	Asymmetric thin film composite	Asymmetric thin film composite
Permeate	Water + dissolved solutes	Water + small molecules	Water + monovalent and some divalent ions	Water
Retentate	Large suspended particles, some emulsion, most bacteria	Most organics over 1000 molecular weight including pyrogenes, viruses, bacteria and colloids	Salts, organic molecules over 500	Ions and most organics over 200 molecular weight

The primary role of a membrane is to act as a selective barrier. It should permit passage of certain components and retain certain other components of a mixture. Membrane is a phase that acts as a barrier to prevent mass movement but allowing restricted and/or regulated passage of one or more species through it (Lakshminarayanaiah, 1984). In the dairy industry, membrane processes used are microfiltration, ultrafiltration, nanofiltration and reverse osmosis. The use of reverse osmosis and ultrafiltration was first started in 1960 for desalination and water treatment. The process gained entrance in food industry, particularly the dairy, in the seventies and its application increased dramatically in the 1980s. The first pilot studies on microfiltraion of whole milk were reported in 1987 while the nanofiltration has been in use in dairy industry only very recently. What distinguishes the membrane processes is the application of hydraulic pressure to speed up the transport processes. The nature of the membrane controls which component permeated and which component is retained. Reverse osmosis is a dewatering technique while ultrafiltration is a method for purifying, concentrating and fractionating macromolecules or fine colloidal suspensions. Microfiltration is a loose ultrafiltration and allows separation of suspended particles, i.e. fat globules, microorganisms’ etc. from dissolved substances in a feed stream. The principle application of nanofiltration is in partial demineralization of whey. The membranes are made up of different types of materials-naturally available materials such as cellulose, or synthetic polymers such as polycarbonate, polyethylene, polysulfone, or modifications of existing polymers, such as sulfonated polysulfones or entirely new polymers developed specifically for membrane application. Most membranes are manufactured in the “flat sheet” form and in some cases they may be cast directly on to the module itself e.g. in tubular modules or may be extruded or spun into hollow fibers directly. In India, the use of membranes presents several limitations such as high initial capital cost of the equipment installation, lack of indigenous backup support for replenishment of membranes, inadequate technological expertise of the plant operators, fouling of the membranes, etc.

Membrane Technology- Applications in Dairy Industry

The primary role of a membrane is to act as a selective barrier. It should permit passage of certain components and retain certain other components of a mixture. Membrane is a phase that acts as a barrier to prevent mass movement but allowing restricted and/or regulated passage of one or more species through it . In the dairy industry, membrane processes used are microfiltration, ultrafiltration, nanofiltration and reverse osmosis. The use of reverse osmosis and ultrafiltration was first started in 1960 for desalination and water treatment. The process gained entrance in food industry, particularly the dairy, in the seventies and its application increased dramatically in the 1980s. The first pilot studies on microfiltraion of whole milk were reported in 1987 while the nanofiltration has been in use in dairy industry only very recently. What distinguishes the membrane processes is the application of hydraulic pressure to speed up the transport processes. The nature of the membrane controls which component permeated and which component is retained. Reverse osmosis is a dewatering technique while ultrafiltration is a method for purifying, concentrating and fractionating macromolecules or fine colloidal suspensions. Microfiltration is a loose ultrafiltration and allows separation of suspended particles, i.e. fat globules, microorganisms’ etc. from dissolved substances in a feed stream. The principle application of nanofiltration is in partial demineralization of whey. The membranes are made up of different types of materials-naturally available materials such as cellulose, or synthetic polymers such as polycarbonate, polyethylene, polysulfone, or modifications of existing polymers, such as sulfonated polysulfones or entirely new polymers developed specifically for membrane application. Most membranes are manufactured in the “flat sheet” form and in some cases they may be cast directly on to the module itself e.g. in tubular modules or may be extruded or spun into hollow fibers directly. In India, the use of membranes presents several limitations such as high initial capital cost of the equipment installation, lack of indigenous backup support for replenishment of membranes, inadequate technological expertise of the plant operators, fouling of the membranes, etc.

pH test for milk testing

     

A.     USING pH PAPER

Certain indicators show change in colour with the change in pH. The pH paper or strips are impregnated with these indicators such as bromothymol blue (pH 6.0 to 7.6) and bromocresol purple (pH 5.2 to 6.8). pH papers in narrow range and wide range are available.

Objective

·         To study the freshness of milk.

Reagents

·         pH paper strips ranges 4-10

Procedure

·         Take a small quantity of milk in the test tube.

·         Dip the pH strip in the milk.

·         Compare the colour changes with standard chart and note the pH.

Precaution

·         The results with pH paper are not precise and for more precision pH meter is used.

·         The pH strips should be stored in a glass bottle properly stoppered in dry conditions.

·         The electrode of the pH meter when dipped in the sample, detect the active hydrogen ions and the    potential difference is measured in terms of pH.

B.      USING pH METER

Objectives:

·         To study the freshness of milk.

·         To detect mastitis milk or neutralised milk.

Apparatus

·         pH meter(Pocket).

·         Beakers - 100 ml capacity.

·         Tissue paper/filter paper.

·         Wash bottle containing distilled water.

Reagents

·         Buffer solution of pH 4.0, 7.0 and 9.2.

Procedure

•          Switch on the pocket pH meter as per instruction of manufacturer.

•          Wash the electrodes with distilled water.

•          Dry the electrodes with the help of tissue paper/filter paper.

•          Take buffer solution in a beaker and dip the electrodes in it.

•          Adjust the pH meter to the pH of buffer solution.

•          Mix the milk sample thoroughly and take in a beaker.

•          Dip the washed and dry electrode in it and observe the reading on the scale.

•          Normal milk pH is between 6.6-6.8. pH above 6.9 is indication of mastitic milk/late lactation milk and pH lower than 6.5 means developed acidity.

Alcohol Alizarin test

Developed acidity makes the milk unstable to heat.
Objective:
• To assess the suitability of milk for high heat treatment and also to get idea about the acidity of milk.
Apparatus
• Test tube
Reagents
• Alcohol alizarine solution - 0.2 per cent
Procedure
• Take about 5 ml of milk in a test tube.
• Add equal amount of alcohol alizarin solution (0.2%).
• Mix the contents well.
• Observe for presence of flakes and colour of the contents.
Range of colour Presence of flakes Approx acidity (% LA) Heat stability
Lilac or pale red No flakes o.14 Good (low acidity)
Lilac or pale red Small flakes 0.14—0.17 Poor (sweet curdling)
Violet or purple (alkaline) Small flakes 0.17 Poor (late lacta¬tion or mastitis)
Reddish brown (acidic) Small flakes 0.17-0.20 Very poor
Brownish yellow to yellow (highly acidic) Large flakes 0.20 Very poor

Alcohol test

     The salts of milk are in a particular ratio with each other. When this ratio is disturbed because of colostrum, late lactation or udder infection, milk becomes less stable to heat.

0bjectives:

·         To study the stability of milk to processing particularly for condensing and sterilization.

·         To detect abnormal milk such as colostrum, late lactation, mastitic milk and milk with salt imbalance.

Apparatus

·         Test tube preferably with a mark at 5 and 10 ml

Reagents

·         Ethyl Alcohol: 68 % (w/v) or 75% (v/v)

Procedure

·         Transfer 5 ml of milk sample to a test tube and add equal quantity of (75% by vol.) ethyl alcohol.

·         Mix the contents of the test tube by inverting several times.

·         Examine for the presence of flakes or clots and also note the size of flakes.

Clot-on-Boiling (COB) Test

The development of acid in milk by conversion of lactose to lactic acid makes the milk unstable to heat and milk coagulates on boiling. Generally, milk samples with more than 0.20% acidity give a positive COB test.
Objectives
• To determine developed acidity
• To know the heat stability for processing of milk
Apparatus
• Test tube preferably with a mark at 5 ml
• Test tube holder
• Boiling water bath
Procedure
• Transfer 5 ml of milk into the test tube.
• Place the test tube in a water bath at boiling temperature for 5 to 6 minutes.
• Remove the tube from the bath and rotate them in an almost horizontal position.
• Examine the film of milk on the sides of the test tube for any precipitated particles.
• The formation of clot or presence of precipitated particles constitutes a positive C.O.B. test.

Sediment Tests

 

Milk can be scored for sediment either by observing the particles of sediment which may have settled at the bottom of the bottle or by observing the sediment collected on a filter disc.

Objective:

·         To assess the cleanliness of milk received

Apparatus

·         Sediment tester

·         Sediment discs

·         Standard sediment discs

Procedure

A. Off-bottom sediment test

• Insert sediment pad in the space provided. Dip the tester in milk up to bottom without disturbing the milk.
• By pulling out the plunger collect milk slowly from different parts of the bottom. Later, press the plunger down.
• Remove the sediment pad and compare with the standard sediment disc pads provided or find out the weight of sediment and Compare the sediment disc with the prepared standard sediment discs and record the sediment score.

B. Mixed sample sediment test

• Insert sediment pad in the mixed sample sediment tester.
• Pass a known quantity of the milk (500 ml) through the pad.
• Remove the pad from tester.
• Compare the sediment pad with the standard pad provided or find out weight of sediment and grade the milk according to I.S.I. standards as given below: -

              Amount of sediment in disc (mg)            Quality of milk

0.0                                                                   Excellent

              0.2                                                                 Good

              0.5                                                                 Fair   

              1.0                                                                 Bad

              2.0                                                                 Very bad

Title: SAMPLING OF MILK AND MILK PRODUCTS

Quantity of sample to be collected for analysis
Product	Approximate quantity
Milk	250 ml
Ghee	150g
Butter	150g
Khoa	250g
Dahi	200g
Cream	250g
Ice-cream	300g

It is essential to draw a rep­resentative sample of milk and milk pro­ducts for chemical and microbiological examination to ensure that the product meets the prescribed standards of quality and acceptance. Analysis of milk and milk pro­ducts is carried out for de­termining the various milk constituents and studying its physico-chemical properties. Accurate sampling is most important in all analysis. Seri­ous errors in experimental re­sults are caused due to improper sampling procedures. A sample must be a proper rep­resentative of the lot from which it is collected. Hence, thorough mixing of milk is a must before the collection of a sample. However, vigorous shaking must be avoided as this will cause churning of fat and foaming of milk, etc. A sample must have enough quantity for all types of analysis as given in table for milk and milk products.

Aim

To become familiar with the different procedures to be followed in collecting samples of milk and milk products for analysis.

Procedure

A -Sampling of milk

(i) Sampling from individual container

·         Pour milk from one con­tainer to another 3 or 4 times.

·         When this is not possible, thoroughly mix the milk with a plunger, so that any milk fat adhering to the inner side of the can gets well mixed with the re­mainder of the milk.

·         Collect the sample immediately with a dipper from several spots.

(ii) Sample from individual animal milk

           ·           When the sample of milk is collected from individual animals ensure that the last strippings are drawn from the udder.

           ·           The entire amount of milk is mixed thoroughly the sample col­lected with the help of a dip­per.

           ·           For normal laboratory analysis, about 250 to 500 ml of milk may be collecte­d in clean, dry sample bottles.

 (iii) Sampling from several containers:

Pour all the milk from all the containers into a single vat.

           ·           Mix thoroughly with the help of a plunger.

           ·           Collect the sample with a dipper from separate places.

           ·           If pouring of milk in a vat is not possible then distri­bute milk as equally as possible amongst all the containers.

           ·           Thoroughly mix the milk of all the containers.

           ·           Collect a proportionate volume of milk from each container with a dipper in another vessel.

           ·           After thorough mixing, collect the final sample.

(iv) Sampling from milk storage tanks and road or rail milk tanker

           ·           Thoroughly mix the milk in the tank using a mechanical agitator or a long plunger.

           ·           Collect sample either through the sampling cock or using a dipper or opening the manhole.

(v) Sampling bulk units

           ·           When milk of uniform qual­ity is supplied in bulk units e.g. cans, storage tanks, etc., the number of random units to be drawn shall be as follows:

Total No. of units	No. of units to be selected
1	1
2-5	2
6-20	3
21-60	4
61-100	5
over 100	5 plus one for each additi­onal l00 units or fraction thereof.

(vi) Composite milk samples fat and SNF test

           ·           The suppliers of milk are normally paid on the basis of fat and SNF content of milk. Daily analysis of milk for fat and SNF is time-consuming and quite expensive. In such a case, composite samples of the suppliers' milk are ta­ken.

           ·           The volume of the indi­vidual composite sample should not be less than 150 ml.

           ·           The composite milk sample should be collected during the agreed period by placing into the composite sample bottle, a representa­tive sample proportionate to the amount of milk supplied each day.

           ·           The composite sample should be preserved with 0.1 ml addition of 36% Formaldehyde (Formalin) for every 25 ml of milk.

           ·           The bottle should be tightly stoppered to prevent evaporation and kept away from light.

           ·            It should be tested on the same day as the last portion of milk is collected into the sample bottle.

           ·            After each addition of milk the contents of the sample bottle ate mixed by gentle rotation of the bottle

Treatment of milk on arrival at the laboratory before analysis

           ·           Warm the sample in the bottle to about 40°C in a bath and mix thoroughly. Cool to 26°-28°C. Leave aside the sample for about 4 minutes. After that mix the sample by inverting the bottle 3—4 times and start analysis

B- Sampling of cream

           ·           Mix fresh cream in the can with the help of a plunger or by using a mechanical agitator for not less than 10 minutes. Aged cream may be warmed to 40°C before mixing.

           ·           Collect the sample with the help of a dipper or through a sample cork.

C- Sampling of khoa, chhana and burfi

           ·           Keep the product on a clean flat surface over a        butter paper.

           ·           Remove the top surface of the product by scrap­ping with the help of a, knife or spatula.

           ·           Insert the sampler in the product and rotate it.

           ·           Remove the sampler and transfer the product in a sample bottle.

           ·           Take samples of these products from several places of the same lot and mix to obtain a repre­sentative sample.

D- Sampling of butter

           ·           Remove 3 or 4 cores with a butter trier from various parts of the butter sample.

           ·           Combine the collected cores of butter samples in a bottle with wide mouth.

           ·           Melt the product at 40°C in a water bath by a swirl­ing action and collect the sample.

E- Sampling of Ice-cream

           ·           Take the cup of ice-cream and remove a core from the top.

           ·           Transfer the product to a bottle with a wide mouth.

           ·           Keep the bottle in a water bath at 40°C.

           ·           Mix the product by shaking the bottle slowly.

           ·           Collect the sample after removing the foam completely.

Precautions

·         The sample bottle used should be wide mouthed with sloping sides and should have well fitting caps.

·          The sample bottle should be of such a size that when the required quantity of sample is put into it there should not be much space after putting the stopper. Only rubber stopper should be properly labelled for its content.

·          When mercuric chloride is used for freezing point determination the bottle should be labelled clearly as “Poison”. Mer­curic chloride is to be added at 0.5 gm for each 250 ml milk

           ·           Avoid excessive agitation of milk.

           ·           Prevent contamination of sterilized apparatus.

           ·           Avoid over agitation of cream as this will cause churning of cream.

           ·           When a sample is collected for bacteriological analysis, ensure that the equipment used has been properly sterilized and the sampling is carried out under strict aseptic conditions. Follow the instructions given.

           ·           If small clots or lumps are observed in the sample which cannot be dispersed, a few drops of liquor ammonia may be used during mixing/homogenisation.

           ·           If even after mixing /homogenisation the sample shows lumps or clots or droplets of oil are visible suggestive of curdling /splitting of milk, the sample should be deemed unfit for analysis and rejected.